VIP-related peptides [VIP-related peptides [VIP, PACAP] have widespread effects in the gastrointestinal tract (GI) and central nervous system affecting such diverse functions as motility, secretion and growth of normal and neoplastic tissues. The actions of VIP-PACAP-related peptides is mediated by three receptors (VIP-1-R, VIP-2-R, PACAP-R). Studies in this project are aimed at understanding the molecular basis of action of these different receptors and at developing selective metabolically stable ligands that function as agonists or antagonists at these different receptors. In general, two groups of studies are being performed in this project. To define the pharmacology of VIP receptors, the rat and human VIP-1-R and VIP-2-R receptors have been cloned and stably transfected in CHO and PANC1 cells. Furthermore, various native cells were screened by RT-PCR, Northern blots and binding studies to identify cell lines that have only one native VIP-R subtype. The breast cancer cell line, T47D was found to possess predominantly hVIP-1-R and SupT1 lymphoblasts only hVIP-2-R and to possess sufficient numbers of receptors to be useful for pharmacology and cell biology studies. The VIP pharmacophore is being determined for each VIP-R subtype by alanine and D-amino acid screening, to attempt to identify selective ligands. It has now been completed for the rat and human VIP-1-R and results were published this year. Studies defining the VIP pharmacophore of the human and rat VIP-2-R are in progress. The analysis of the VIP-1-R demonstrates positions 2,8,9,11,19,24,25,27,28 of VIP can be replaced by alanine with no loss of potency. Furthermore, there are significant differences for the VIP pharmacophore for the VIP-1-R in human, rat and guinea pig that have not been considered in previous analogue design studies. A simplified analogue of VIP with 11 alanines with potency equal to VIP has been made and was found to be metabolically stable. This simplified VIP analogue will be an excellent template for future studies. From these studies simplified selective ligands for the hVIP-1-R and hVIP-2-R can be designed. Secondly, the molecular basis for ligand internalization and the role of cellular cyclic AMP in its mediation by VIP related receptors, is being studied. Receptor mutants with varying lengths of COOH terminus deletions have been made. These studies demonstrate even though the VIP-1-R and VIP-2-R are closely related in their C00H terminus, serial truncations have differing effects on internalization and activation of the 2 receptors. Further investigation of these differences will help define the relative importance of this receptor region in mediating these differing effects in the two receptors.